升麻素与SPR结合以非酶方式调节其蛋白表达进而抑制2型气道炎症
2023/03/21
摘要
背景：升麻素是著名中药玉屏风散的主要生物活性成分之一，可有效缓解过敏性哮喘（allergic asthma，AA）和特应性皮炎，减少临床复发。然而，升麻素治疗AA的潜在机制仍然未知。
目的：本研究旨在探讨升麻素对过敏性哮喘的作用和机制。
方法：本研究包括体内和体外实验。体内外实验观察升麻素对AA的影响。预测墨蝶呤还原酶（sepiapterin reductase，SPR）为升麻素通过反向对接治疗AA的最有效靶点。使用分子对接和微型热泳法（microscale thermophoresis，MST）分析升麻素和SPR之间的直接结合。对SPR进行过表达和干扰以验证靶向SPR是否是升麻素治疗AA的关键步骤。在体内和体外加用SPR抑制剂QM385，以评估SPR在AA中的作用。此外，采用高效液相色谱法和无细胞直接hSPR酶活性测定法研究升麻素是否通过影响酶活性来调节SPR。同时，在体外使用蛋白质降解抑制剂来探索升麻素对SPR的作用机制。
结果：本研究发现升麻素通过降低气道高反应性、抑制2型细胞因子介导的气道炎症和恢复上皮屏障蛋白的表达，有效缓解AA。分子对接预测了升麻素与SPR的直接结合能力，MST进一步验证了这一点。值得注意的是，升麻素对AA的治疗作用因SPR干扰而减弱，相反，QM385在体内和体外均显著减轻AA的表型特征。有趣的是，升麻素对SPR的酶活性没有影响，因为通过无细胞酶活性测定，升麻素处理不影响其底物海泡蛋白的水平。此外，本研究发现升麻素可能通过自噬体途径降低SPR蛋白的表达，而不影响其mRNA的表达。
结论：据了解，本研究首次报道升麻素可以通过直接与SPR结合并以非酶的方式调节其蛋白表达来抑制2型气道炎症，从而缓解过敏性哮喘。
Cimifugin suppresses type 2 airway inflammation by binding to SPR and regulating its protein expression in a non-enzymatic manner
Gu X, Chen Y, Qian P, He T, Wu Y, Lin W, Zheng J, Hong M.
Abstract
BACKGROUND:Cimifugin is one of the main bioactive components of Yu-Ping-Feng-San, a well-known traditional Chinese medicine, which can effectively relieve Allergic asthma (AA) and atopic dermatitis and reduce recurrence in clinic. However, the underlying mechanism of cimifugin on AA is still unknown.
OBJECTIVES: In the present study, we aimed to investigate the effect and mechanism of cimifugin on AA.
METHODS:In vivo and in vitro experimental studies were performed. The effect of cimifugin on AA was demonstrated in vivo and in vitro. Sepiapterin reductase (SPR) was predicted as the most potent target of cimifugin in treating AA by reverse docking. Molecular docking and microscale thermophoresis (MST) were used to analyze the direct binding between cimifugin and SPR. Overexpression and interference of SPR were performed to verify whether targeting SPR is a key step of cimifugin in the treatment of AA. QM385, an inhibitor of SPR, was administrated in vivo and in vitro to evaluate the role of SPR in AA. Further, HPLC and cell-free direct hSPR enzyme activity assay were performed to research whether cimifugin regulated SPR by influencing the enzyme activity. Simultaneously, the inhibitors of protein degradation were used in vitro to explore the mechanism of cimifugin on SPR.
RESULTS:We found cimifugin effectively alleviated AA by reducing airway hyperresponsiveness, inhibiting type 2 cytokines-mediated airway inflammation, and restoring the expression of epithelial barrier proteins. Molecular docking predicted the direct binding ability of cimifugin to SPR, which was further verified by MST. Notably, the therapeutic effect of cimifugin on AA was dampened with SPR interfering, in contrast, the phenotypic features of AA were significantly alleviated with QM385 application both in vivo and in vitro. Interestingly, cimifugin showed no effect on the enzyme activity of SPR, as the level of its substrate sepiapterin was not affected with cimifugin treatment by cell-free enzyme activity assay. Furthermore, we found cimifugin could reduce SPR protein expression without affecting its mRNA expression probably through autophagosome pathway.
CONCLUSIONS:To our knowledge, we're reporting for the first time that cimifugin can suppresses type 2 airway inflammation to alleviate AA by directly binding to SPR and regulating its protein expression in a non-enzymatic manner.
背景：升麻素是著名中药玉屏风散的主要生物活性成分之一，可有效缓解过敏性哮喘（allergic asthma，AA）和特应性皮炎，减少临床复发。然而，升麻素治疗AA的潜在机制仍然未知。
目的：本研究旨在探讨升麻素对过敏性哮喘的作用和机制。
方法：本研究包括体内和体外实验。体内外实验观察升麻素对AA的影响。预测墨蝶呤还原酶（sepiapterin reductase，SPR）为升麻素通过反向对接治疗AA的最有效靶点。使用分子对接和微型热泳法（microscale thermophoresis，MST）分析升麻素和SPR之间的直接结合。对SPR进行过表达和干扰以验证靶向SPR是否是升麻素治疗AA的关键步骤。在体内和体外加用SPR抑制剂QM385，以评估SPR在AA中的作用。此外，采用高效液相色谱法和无细胞直接hSPR酶活性测定法研究升麻素是否通过影响酶活性来调节SPR。同时，在体外使用蛋白质降解抑制剂来探索升麻素对SPR的作用机制。
结果：本研究发现升麻素通过降低气道高反应性、抑制2型细胞因子介导的气道炎症和恢复上皮屏障蛋白的表达，有效缓解AA。分子对接预测了升麻素与SPR的直接结合能力，MST进一步验证了这一点。值得注意的是，升麻素对AA的治疗作用因SPR干扰而减弱，相反，QM385在体内和体外均显著减轻AA的表型特征。有趣的是，升麻素对SPR的酶活性没有影响，因为通过无细胞酶活性测定，升麻素处理不影响其底物海泡蛋白的水平。此外，本研究发现升麻素可能通过自噬体途径降低SPR蛋白的表达，而不影响其mRNA的表达。
结论：据了解，本研究首次报道升麻素可以通过直接与SPR结合并以非酶的方式调节其蛋白表达来抑制2型气道炎症，从而缓解过敏性哮喘。
(中日友好医院呼吸与危重症医学科 张婧媛 摘译 林江涛 审校)
(Phytomedicine. 2023 Mar;111:154657. doi: 10.1016/j.phymed.2023.154657.)
(Phytomedicine. 2023 Mar;111:154657. doi: 10.1016/j.phymed.2023.154657.)
Cimifugin suppresses type 2 airway inflammation by binding to SPR and regulating its protein expression in a non-enzymatic manner
Gu X, Chen Y, Qian P, He T, Wu Y, Lin W, Zheng J, Hong M.
Abstract
BACKGROUND:Cimifugin is one of the main bioactive components of Yu-Ping-Feng-San, a well-known traditional Chinese medicine, which can effectively relieve Allergic asthma (AA) and atopic dermatitis and reduce recurrence in clinic. However, the underlying mechanism of cimifugin on AA is still unknown.
OBJECTIVES: In the present study, we aimed to investigate the effect and mechanism of cimifugin on AA.
METHODS:In vivo and in vitro experimental studies were performed. The effect of cimifugin on AA was demonstrated in vivo and in vitro. Sepiapterin reductase (SPR) was predicted as the most potent target of cimifugin in treating AA by reverse docking. Molecular docking and microscale thermophoresis (MST) were used to analyze the direct binding between cimifugin and SPR. Overexpression and interference of SPR were performed to verify whether targeting SPR is a key step of cimifugin in the treatment of AA. QM385, an inhibitor of SPR, was administrated in vivo and in vitro to evaluate the role of SPR in AA. Further, HPLC and cell-free direct hSPR enzyme activity assay were performed to research whether cimifugin regulated SPR by influencing the enzyme activity. Simultaneously, the inhibitors of protein degradation were used in vitro to explore the mechanism of cimifugin on SPR.
RESULTS:We found cimifugin effectively alleviated AA by reducing airway hyperresponsiveness, inhibiting type 2 cytokines-mediated airway inflammation, and restoring the expression of epithelial barrier proteins. Molecular docking predicted the direct binding ability of cimifugin to SPR, which was further verified by MST. Notably, the therapeutic effect of cimifugin on AA was dampened with SPR interfering, in contrast, the phenotypic features of AA were significantly alleviated with QM385 application both in vivo and in vitro. Interestingly, cimifugin showed no effect on the enzyme activity of SPR, as the level of its substrate sepiapterin was not affected with cimifugin treatment by cell-free enzyme activity assay. Furthermore, we found cimifugin could reduce SPR protein expression without affecting its mRNA expression probably through autophagosome pathway.
CONCLUSIONS:To our knowledge, we're reporting for the first time that cimifugin can suppresses type 2 airway inflammation to alleviate AA by directly binding to SPR and regulating its protein expression in a non-enzymatic manner.
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度普利尤单抗在有或无过敏性哮喘证据的中度至重度2型哮喘儿童中的评估
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